The Prognostic Value of Sex-Determining Region Y-Box 2 and CD8+ Tumor-Infiltrating Lymphocytes in Limited-Stage Small-Cell Lung Cancer.

BACKGROUND: Sex-determining region Y-box 2 (SOX2) is a transcriptional factor that drives embryonic stem cells to neuroendocrine cells in lung development and is highly expressed in small-cell lung cancer (SCLC). However, the prognostic role of SOX2 and its relationship with tumor-infiltrating lymphocytes (TILs) has not been determined in SCLC. Herein, we assessed the expression of SOX2 and CD8+ TILs to obtain insights into the prognostic role of SOX2 and CD8+ TILs in limited-stage (LS)-SCLC. METHODS: A total of 75 patients with LS-SCLC was enrolled. The SOX2 expression and CD8+ TILs were evaluated by immunohistochemistry. RESULTS: High SOX2 and CD8+ TIL levels were identified in 52 (69.3%) and 40 (53.3%) patients, respectively. High SOX2 expression was correlated with increased density of CD8+ TILs (p = 0.041). Unlike SOX2, high CD8+ TIL numbers were associated with significantly longer progression-free survival (PFS; 13.9 vs. 8.0 months, p = 0.014). Patients with both high SOX2 expression and CD8+ TIL numbers (n = 29, 38.7%) had significantly longer PFS and overall survival (OS) compared to those from the other groups (median PFS 19.3 vs. 8.4 months; p = 0.002 and median OS 35.7 vs. 17.4 months; p = 0.004, respectively). Multivariate Cox regression analysis showed that the combination of high SOX2 expression and CD8+ TIL levels was an independent good prognostic factor for OS (HR = 0.471, 95% CI, 0.250-0.887, p = 0.02) and PFS (HR = 0.447, 95% CI, 0.250-0.801, p = 0.007) in SCLC. CONCLUSIONS: Evaluation of the combination of SOX2 and CD8+ TIL levels may be of a prognostic value in LS-SCLC.

Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer.

The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5' fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.

Negative prognostic effect of low nuclear GLI1 expression in glioblastomas.

The hedgehog signaling plays supportive roles in various aspects of tumorigenesis. Increased expression of the key component, GLI1, has been shown to correlate with poor prognosis in many types of cancers. We aimed to investigate the effect of GLI1 expression in glioblastoma focusing on the nuclear localization. Immunohistochemistry for GLI1, GLI2, PTCH1, SMO, and SHH were done in 140 glioblastoma tissues, and the staining was graded. For GLI1, nuclear and cytoplasmic expression was separately assessed. No significant correlation was found between clinicopathologic parameters and expression grades of the five proteins. Low nuclear GLI1 expression was associated with a worse progression-free survival while overall survival was not significantly affected. In contrast, cytoplasmic GLI1 expression did not have a prognostic effect. PTCH1 expression correlated with nuclear GLI1 expression without exerting a significant prognostic effect. Analysis of the TCGA-glioblastoma dataset revealed that low GLI1 mRNA level also correlated with a poor prognosis for both overall and progression-free survival. The adverse effect of low nuclear GLI1 expression in glioblastomas is in contrast with the negative prognostic effect of high GLI1 expression reported in non-cranial malignancies. The relative impact of hedgehog signaling among other oncogenic pathways in the brain may be responsible for the difference. The different implication of GLI1 expression in glioblastomas needs to be considered in studies of hedgehog signaling-targeted therapy.

Poorly Differentiated Clusters in Colorectal Adenocarcinomas Share Biological Similarities with Micropapillary Patterns as well as Tumor Buds.

In colorectal carcinoma, poorly differentiated clusters (PDCs) are a poor prognostic indicator and show morphological continuity and behavioral similarities to micropapillary patterns (MPPs) as well as tumor buds (TBs). Epithelial-mesenchymal transition (EMT) and inhibition of cancer-stromal interactions may contribute to the development of PDCs. To clarify the biological nature of PDCs, we examined immunohistochemical stainings for ß-catenin, Ki-67, E-cadherin, epithelial cell adhesion molecule (EpCAM), MUC1, and epithelial membrane antigen (EMA), which are associated with EMT and cancer-stromal interactions. The expression frequencies and patterns of PDCs, TBs, and differentiated neoplastic glands from the tumor center (TC) were compared. In the study group (117 cases), the nuclear ß-catenin staining index was higher in PDCs (37.3%) and TBs (43.3%) than in neoplastic glands from TC (8.9%, P < 0.001). The mean Ki-67 labeling index in TC was 71.5%, whereas it was decreased in PDCs (31.2%) and TBs (10.2%, P < 0.001). E-cadherin and EpCAM displayed a tendency to be found along the cell membrane in TC samples (91.5% and 92.3%, respectively), whereas they showed loss of membranous staining in PDC (44.4% and 36.8%, respectively) and TB samples (60.7% and 68.4%, respectively). An inside-out pattern for MUC1 and EMA was frequently observed in PDC (48.7% and 45.3%, respectively) and TB samples (46.2% and 45.3%, respectively), but not in TC samples. Our data demonstrate that there is a pathogenetic overlap among PDCs, TBs, and MPPs and suggest that they might represent sequential growth patterns that branch from common biological processes such as dedifferentiation and alteration in cancer-stromal interactions.

Nasal-type NK/T-cell lymphomas are more frequently T rather than NK lineage based on T-cell receptor gene, RNA, and protein studies: lineage does not predict clinical behavior.

Extranodal natural killer (NK)/T-cell lymphoma (ENKTL), nasal type, comprises NK or cytotoxic T cells. We evaluated the clinical impact of cell type and the usefulness of T-cell receptor (TCR) gene transcripts in distinguishing cell lineage. One hundred and eight cases of ENKTL were analyzed for TCR gene rearrangements using the BIOMED-2 protocol and for TCR gene expression using immunohistochemistry for TCR-ßF1 and TCR-cγM1, and RNA in situ hybridization for TCR gene transcripts. Prognostic factors were analyzed. Among the 108 cases, 44 were monoclonal for a TCR rearrangement (40%) while 64 (60%) were undefinable. The monoclonal cases expressed TCR-ßF1 in 14 out of 40 cases (35%) and TCR-cγM1 in 1 out of 44 cases (2%). The 64 undetermined cases expressed TCR-ßF1 in 15 cases (23%) and TCR-cγM1 in 1 (2%). Thirteen of 40 TCR-ß constant gene transcript-positive cases (33%) expressed TCR-ßF1 and one of nine TCR-γ constant gene transcript-positive cases (11%) expressed TCR-cγM1. TCR gene transcripts were not useful in the distinction of cell lineages. TCR gene transcripts were positive in ENKTLs as well as in normal B cells and aggressive NK-cell leukemia. Based on gene rearrangements and immunohistochemistry for TCR, there were 60 T-cell type cases (56%), 32 NK-cell type cases (30%), and 16 cases with an undetermined cell type (14%). TCR protein was expressed in 30/60 T-ENKTLs (50%) in a variable fraction of tumor cells. There were no significant differences in clinical findings or overall patient survival between T- or NK-cell types of ENKTL, although those with a T-cell type tended to show a better prognosis for those with localized nasal lymphomas. Univariate and multivariate analysis showed that a non-nasal ENKTL, age >60 years, high level of lactate dehydrogenase, bone marrow involvement, and the absence of radiotherapy were independent prognostic factors.

FGFR2 in gastric cancer: protein overexpression predicts gene amplification and high H-index predicts poor survival.

FGFR2 gene amplification, and resulting FGFR2 protein overexpression, is rare in gastric cancer patients, and development of an accurate and widely available method for mass screening to identify patients who may respond to treatment with fibroblast growth factor receptor (FGFR) inhibitors is important. We first screened 312 gastric cancer patients with known copy number variations by FGFR2b immunohistochemistry using FPR2-D, an isoform-specific antibody. Next, we performed immunohistochemistry on tissue microarrays from 1574 gastric cancer patients. Selected cases were analyzed for FGFR2 amplification by FISH. In addition, FGFR2b overexpression was studied in 88 matched primary and metastatic gastric cancers. In the first cohort, FGFR2b immunohistochemistry results correlated very well with those of copy number variation (r=0.79) and FISH (r=1.0). In total, FGFR2b overexpression was identified in 73 of 1974 gastric cancers (4%). The concordance between immunohistochemistry and FISH was extremely high; all 2+ and 3+ cases identified by immunohistochemistry were FGFR2 amplified. In the matched primary and metastatic gastric cancer pairs, the positivity and percentage of positive tumor cells were significantly higher in metastatic gastric cancers than in primary gastric cancers (8% vs 3% and 75% vs 47%, respectively; P<0.001). FGFR2b overexpression was significantly more frequent in gastric cancers with diffuse subtype (P=0.01) and higher N stage (P=0.006). FGFR2b overexpression with H-score ≥150 were independent prognostic factors for overall survival with hazard ratio of 1.836 (95% confidence interval, 1.034-3.261; P=0.038). FGFR2b positivity in immunohistochemistry was strongly correlated with FGFR2 amplification. Given the low frequency of FGFR2 amplification in gastric cancers, FGFRb2 immunohistochemistry is an accurate screening tool to detect FGFR2 amplification, and both primary and metastatic gastric cancer tissues should be tested to select gastric cancer patients for treatment with FGFR2 inhibitors.

Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma.

The success of crizotinib in ALK-positive patients has elicited efforts to find new oncogenic fusions in lung cancer. These efforts have led to the discovery of novel oncogenic fusion genes such as ROS1 and RET. However, the molecular and clinicopathologic characteristics associated with RET or ROS1 fusion, compared with ALK fusion-positive lung cancer, remain unclear. We accordingly analyzed the clinicopathologic characteristics of RET- and ROS1-fusion-positive lung adenocarcinomas. We further performed immunohistochemistry and fluorescence in situ hybridization analysis (FISH) in 15 cases of RET and 9 cases of ROS1 fusion tumors by identified NanoString's nCounter screening. RET fusion-positive patients were younger in age, never-smokers, and in early T stage; ROS1 fusion-positive patients had a higher number of never-smokers compared with patients with quintuple-negative (EGFR-/KRAS-/ALK-/ROS1-/RET-) lung adenocarcinoma. Histologically, RET and ROS1 fusion tumors share the solid signet-ring cell and mucinous cribriform pattern, as previously mentioned in the histology of ALK fusion tumors. Therefore, it can be presumed that fusion gene-associated lung adenocarcinomas share similar histologic features. In immunohistochemistry, the majority of 15 RET and 9 ROS1 fusion-positive cases showed positivity of more than moderate intensity and cytoplasmic staining for RET and ROS1 proteins, respectively. In FISH, the majority of RET and ROS1 rearrangement showed two signal patterns such as one fusion signal and two separated green and orange signals (1F1G1O) and an isolated 3' green signal pattern (1F1G). Our study has provided not only characteristics of fusion gene-associated histologic features but also a proposal for a future screening strategy that will enable clinicians to select cases needed to be checked for ROS1 and RET rearrangements based on clinicohistologic features.

Enhanced autotrophic astaxanthin production from Haematococcus pluvialis under high temperature via heat stress-driven Haber-Weiss reaction.

High temperatures (30-36 °C) inhibited astaxanthin accumulation in Haematococcus pluvialis under photoautotrophic conditions. The depression of carotenogenesis was primarily attributed to excess intracellular less reactive oxygen species (LROS; O2 (-) and H2O2) levels generated under high temperature conditions. Here, we show that the heat stress-driven inefficient astaxanthin production was improved by accelerating the iron-catalyzed Haber-Weiss reaction to convert LROS into more reactive oxygen species (MROS; O2 and OH·), thereby facilitating lipid peroxidation. As a result, during 18 days of photoautotrophic induction, the astaxanthin concentration of cells cultured in high temperatures in the presence of iron (450 µM) was dramatically increased by 75 % (30 °C) and 133 % (36 °C) compared to that of cells exposed to heat stress alone. The heat stress-driven Haber-Weiss reaction will be useful for economically producing astaxanthin by reducing energy cost and enhancing photoautotrophic astaxanthin production, particularly outdoors utilizing natural solar radiation including heat and light for photo-induction of H. pluvialis.

Enhanced astaxanthin production from microalga, Haematococcus pluvialis by two-stage perfusion culture with stepwise light irradiation.

For efficient astaxanthin production from the culture of green microalga, Haematococcus pluvialis, a two-stage mixotrophic culture system was established with stepwise increased light irradiance. By perfusion process, high density biomass (2.47 g/L) was achieved during the vegetative stage due to no detrimental effect of inhibitory metabolites, which was 3.09 and 1.67 times higher than batch and fed-batch processes, respectively. During the induction stage, biomass and astaxanthin were subsequently produced to the very high level 12.3 g/L and 602 mg/L, under stepwise increased light irradiance (150-450 µE/m(2)/s), respectively. These results indicate that the combinatorial approach of perfusion culture during the vegetative stage and stepwise light irradiation during the induction stage is a promising strategy for the simultaneous production of high concentration of biomass and astaxanthin in microalgae including H. pluvialis.

Madecassoside modulates lipid metabolism in visceral adipocytes: exploring the browning, lipolysis, and lipogenesis mechanisms for potential obesity treatment.

OBJECTIVES: Madecassoside (MA) is a triterpene derived from Centella asiatica that has been recognized for its antioxidant and anti-inflammatory properties in various disease models. However, its direct impact on cultured white adipocytes and the underlying mechanisms, mainly through gene knockdown, have not been thoroughly explored. METHODS: Western blot analysis was utilized to assess the expression levels of various proteins, while oil red O staining was used to measure lipid deposition. The adipocyte shapes were confirmed using H&E staining. KEY FINDINGS: MA treatment enhanced browning and lipolysis in 3T3-L1 adipocytes and adipose tissue from experimental mice while suppressing lipogenesis. Furthermore, MA treatment increased the expression of PPARα and FGF21 in 3T3-L1 adipocytes as well as the secretion of FGF21 into the culture medium. Knockdown of PPARα or FGF21 using siRNA diminished the effects of MA on lipid metabolism in cultured adipocytes. CONCLUSIONS: These findings demonstrate that MA promotes thermogenic browning and lipolysis while inhibiting adipocyte lipogenesis, thus showing the potential for attenuating obesity. The study suggested that MA could be a viable therapeutic approach for treating obesity.

Madecassoside ameliorates hepatic steatosis in high-fat diet-fed mice through AMPK/autophagy-mediated suppression of ER stress.

Hepatic endoplasmic reticulum (ER) stress is a contributing factor in the development of hepatic steatosis in obesity. Madecassoside (MA), a pentacyclic triterpene derived from Centella asiatica, is known for its anti-inflammatory properties in the treatment of skin wounds. However, the impact of MA on hepatic ER stress and lipid metabolism in experimental obesity models has not been investigated. In this study, we examined the effects of MA on primary hepatocytes treated with palmitate and the livers of mice fed a high-fat diet (HFD). Our findings demonstrated that MA treatment reduced lipogenic lipid accumulation, apoptosis, and ER stress in hepatocytes. Additionally, MA treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and markers of autophagy. Importantly, when AMPK was inhibited by small interfering RNA (siRNA) or autophagy was blocked by 3-methyladenine (3MA), the protective effects of MA against ER stress, lipogenic lipid deposition, and apoptosis in palmitate-treated hepatocytes were abolished. These results suggest that MA mitigates hepatic steatosis in obesity through an AMPK/autophagy-dependent pathway. The present study highlights the potential of MA as a promising therapeutic candidate for hepatic steatosis.

miR-199a and miR-199b facilitate diffuse gastric cancer progression by targeting Frizzled-6.

Pathological markers that can monitor the progression of gastric cancer (GC) may facilitate the diagnosis and treatment of patients with diffuse GC (DGC). To identify microRNAs (miRNAs) that can differentiate between early and advanced DGC in the gastric mucosa, miRNA expression profiling was performed using the NanoString nCounter method in human DGC tumors. Ectopic expression of miR-199a and miR-199b (miR-199a/b) in SNU601 human GC cells accelerated the growth rate, viability, and motility of cancer cells and increased the tumor volume and weight in a mouse xenograft model. To study their clinicopathological roles in patients with GC, miR-199a/b levels were measured in human GC tumor samples using in situ hybridization. High miR-199a/b expression level was associated with enhanced lymphovascular invasion, advanced T stage, and lymph-node metastasis. Using the 3'-untranslated region (UTR) luciferase assay, Frizzled-6 (FZD6) was confirmed to be a direct target of miR-199a/b in GC cells. siRNA-mediated depletion of FZD6 enhanced the motility of SNU601 cells, and addback of FZD6 restored cancer cell motility stimulated by miR-199a/b. In conclusion, miR-199a/b promotes DGC progression by targeting FZD6, implying that miR-199a/b can be used as prognostic and diagnostic biomarkers for the disease.

Automated Diagnosis of Cervical Intraepithelial Neoplasia in Histology Images via Deep Learning.

Artificial intelligence has enabled the automated diagnosis of several cancer types. We aimed to develop and validate deep learning models that automatically classify cervical intraepithelial neoplasia (CIN) based on histological images. Microscopic images of CIN3, CIN2, CIN1, and non-neoplasm were obtained. The performances of two pre-trained convolutional neural network (CNN) models adopting DenseNet-161 and EfficientNet-B7 architectures were evaluated and compared with those of pathologists. The dataset comprised 1106 images from 588 patients; images of 10% of patients were included in the test dataset. The mean accuracies for the four-class classification were 88.5% (95% confidence interval [CI], 86.3-90.6%) by DenseNet-161 and 89.5% (95% CI, 83.3-95.7%) by EfficientNet-B7, which were similar to human performance (93.2% and 89.7%). The mean per-class area under the receiver operating characteristic curve values by EfficientNet-B7 were 0.996, 0.990, 0.971, and 0.956 in the non-neoplasm, CIN3, CIN1, and CIN2 groups, respectively. The class activation map detected the diagnostic area for CIN lesions. In the three-class classification of CIN2 and CIN3 as one group, the mean accuracies of DenseNet-161 and EfficientNet-B7 increased to 91.4% (95% CI, 88.8-94.0%), and 92.6% (95% CI, 90.4-94.9%), respectively. CNN-based deep learning is a promising tool for diagnosing CIN lesions on digital histological images.

Fluid shear stress facilitates prostate cancer metastasis through Piezo1-Src-YAP axis.

AIMS: Mechanical forces surrounding solid tumors are pervasive in the tumor microenvironment (TME) and abnormally altered as solid tumors progress. Although it has been reported that biomechanical forces, including wall shear stress (WSS), enhance the metastatic features of cancer cells, its mechanism remains unknown. Here, we investigate how cancer cells sense mechanical stress and propagate signals in the TME. MAIN METHODS: Using a microfluidic device, interstitial fluid-mimicking flow (0.05 dyne cm-2) was applied to the human prostate cancer cell line PC3. Piezo1 siRNA and shRNA lentivirus were applied to PC3 cells to ablate Piezo1 expression. PC3-Luc2 cells expressing control shRNA or shPiezo1 lentivirus were administered into the prostate of BALB/c mice for orthotopic injection. KEY FINDING: Here, we show that Piezo1, a mechanosensitive ion channel, is activated by WSS in microfluidic channels. Moreover, Yoda1, a Piezo1 agonist, synergistically potentiates cancer cell motility and nuclear retention of YAP/TAZ via WSS. Also, Piezo1 increases Src phosphorylation, which activates YAP. Conversely, silencing Piezo1 significantly reduces cell motility and YAP/TAZ activity induced by WSS, and finally retards tumor growth and metastasis of administered PC3 cells in BALB/c mice. SIGNIFICANCE: Taken together, these results demonstrate that Piezo1 allows cancer cells to sense mechanical stimuli by altering the microenvironment during tumor progression and is a critical player in modulating cancer metastasis through the Piezo1-Src-YAP axis.

Prognostic value of Dickkopf-1 and ß-catenin expression according to the antitumor immunity of CD8-positive tumor-infiltrating lymphocytes in biliary tract cancer.

The role of ß-catenin and Dickkopf-1 (DKK1) is dependent on the specific immunobiology of T cell inflammation in biliary tract cancer (BTC). We aimed to analyze the role of DKK1 or ß-catenin as a prognostic factor in BTC, and determine the clinical associations of ß-catenin and DKK1 with CD8+ tumor-infiltrating lymphocytes (TIL). We used data from The Cancer Genome Atlas Research Network and the clinicopathological data of 145 patients with BTC who had undergone primary radical resection between 2006 and 2016. CD8+ TIL expression was a significant predictor of favorable overall survival (OS) and relapse-free survival (RFS) (median OS, 34.9 months in high-TIL, 16.7 months in low-TIL, P < 0.0001 respectively; median RFS, 27.1 months in high-TIL, 10.0 months in low-TIL, P < 0.0001 respectively). In the high-CD8+ TIL BTC group, the tumor expression of ß-catenin and DKK1 had a significant negative impact on either OS or RFS. In the low-TIL BTC group, there were no differences according to ß-catenin and DKK1 expression. Cox regression multivariate analysis demonstrated that CD8+ TIL and ß-catenin retained significant association with OS. Among patients with resected BTC, the ß-catenin and DKK1 protein and high CD8+ TIL levels were associated with poor and good clinical outcomes, respectively.

Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering.

Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.

Negative Prognostic Implication of TERT Promoter Mutations in Human Papillomavirus-Negative Tonsillar Squamous Cell Carcinoma Under the New 8th AJCC Staging System.

Telomerase reverse transcriptase gene promoter (TERTp) mutation is a potential candidate for pathogenesis and therapeutic target of tonsillar squamous cell carcinomas (TSCCs) in association with human papillomavirus (HPV). Their clinical relevance has not been validated under the new 8th American Joint Committee on Cancer (AJCC) staging system. We analyzed real-time peptide nucleic acid-mediated PCR and sequencing methods (TERTp mutation) and real-time PCR-based assay (HPV) in 80 surgically resected TSCCs. The 8th edition staging system improved the stratification of the early and advanced stages and between T or N categories for overall survival over the 7th edition. TERTp mutation was found in 7.5%, and HPV in 80.0% of the patients. The majority (83.3%) of TERTp mutation cases were HPV-positive TSCCs. Applying the 8th edition staging system, TERTp mutation was an independent factor of poor prognosis for disease-free survival (DFS) in TSCC patients, supporting the clinical significance of TERTp mutation in tonsil cancer. TERTp mutations were also negatively correlated with overall survival and DFS in HPV-negative TSCCs. Conclusively, TERTp mutation provides negative prognostic impact on survival of surgically managed tonsil cancers staged with the AJCC 8th edition.

Restoration of growth phenotypes of Escherichia coli DH5alpha in minimal media through reversal of a point mutation in purB.

A point mutation (E115K) resulting in slower growth of Escherichia coli DH5alpha and XL1-Blue in minimal media was identified in the purB gene, coding for adenylosuccinate lyase (ASL), through complementation with an E. coli K-12 genomic library and serial subcultures. Chromosomal modification reversing the mutation to the wild type restored growth phenotypes in minimal media.

Programmed cell death-ligand 1 expression in stromal immune cells is a marker of breast cancer outcome.

Purpose: The programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis plays an important role in antitumor immune responses. However, there is considerable inconsistency regarding the prognostic value of PD-L1 expression status in breast cancer. We sought to evaluate the differential prognostic impacts of tumoral versus stromal immune cell PD-L1 expression in primary breast cancer. Materials & Methods: Both tumoral and stromal immune PD-L1 expression in formalin-fixed, paraffin-embedded tumor samples from 233 breast cancer patients without initial stage IV metastases were evaluated by immunohistochemistry using a mouse monoclonal anti-PDL1 antibody. Clinicopathological variables were also documented. A Cox regression model was used to assess the association of tumoral/stromal immune PD-L1 expression with clinical outcome using disease-free survival (DFS) as the primary end point. Results: Both tumoral and stromal immune PD-L1 expression were associated with aggressive tumor characteristics, including higher histologic grade, as well as negative estrogen receptor, negative progesterone receptor, and positive human epithelial growth factor receptor 2 (HER2) status Multivariate analyses further demonstrated that stromal immune cell, but not tumoral, PD-L1 expression was a favorable prognostic factor for survival. Conclusions: Despite its association with aggressive tumor features, PD-L1 expression on stromal immune cells emerged as a positive prognostic biomarker in breast cancer. This pro-survival effect might reflect the presence of a strong antitumor immune response that leads to PD-L1 expression.